Heather Zhou

Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease. To elucidate the molecular basis of NAFLD, we performed an exome-wide association study of liver fat content. Three variants were associated with higher liver fat levels at the exome-wide significance level of 3.6 × 10−7: two in PNPLA3, an established locus for NAFLD, and one (encoding p.Glu167Lys) in TM6SF2, a gene of unknown function. The TM6SF2 variant encoding p.Glu167Lys was also associated with higher circulating levels of alanine transaminase, a marker of liver injury, and with lower levels of low-density lipoprotein–cholesterol (LDL-C), triglycerides and alkaline phosphatase in 3 independent populations (n > 80,000). When recombinant protein was expressed in cultured hepatocytes, 50% less Glu167Lys TM6SF2 protein was produced relative to wild-type TM6SF2. Adeno-associated virus–mediated short hairpin RNA knockdown of Tm6sf2 in mice increased liver triglyceride content by threefold and decreased very-low-density lipoprotein (VLDL) secretion by 50%. Taken together, these data indicate that TM6SF2 activity is required for normal VLDL secretion and that impaired TM6SF2 function causally contributes to NAFLD.

Suppression of PC-1/ENPP-1 expression improves insulin sensitivity in vitro and in vivo

Plasma cell membrane glycoprotein-1, or ectonucleotide pyrophosphatase/phosphodieterase (PC-1/ENPP1) has been shown to inhibit insulin signaling in cultured cells in vitro and in transgenic mice in vivo when overexpressed. Furthermore, both genetic polymorphism and increased expression of PC-1 have been reported to be associated with type 2 diabetes in humans. Thus it was proposed that PC-1 inhibition represents a potential strategy for the treatment of type 2 diabetes. However, it has not been proven that suppression of PC-1 expression or inhibition of its function will actually improve insulin sensitivity. We show in the current study that transient overexpression of PC-1 inhibits insulin-stimulated insulin receptor tyrosine phosphorylation in HEK293 cells, while knockdown of PC-1 with siRNA significantly increases insulin-stimulated Akt phosphorylation in HuH7 human hepatoma cells. Adenoviral vector expressing a short hairpin RNA against mouse PC-1 (PC-1shRNA) was utilized to efficiently knockdown PC-1 expression in the livers of db/db mice. In comparison with db/db mice treated with a control virus, db/db mice treated with the PC-1shRNA adenovirus had approximately 80% lower hepatic PC-1 mRNA levels, approximately 30% lower ambient fed plasma glucose, approximately 25% lower fasting plasma glucose, and significantly improved oral glucose tolerance. Taken together, these results demonstrate that suppression of PC-1 expression improves insulin sensitivity in vitro and in an animal model of diabetes, supporting the proposition that PC-1 inhibition is a potential therapeutic approach for the treatment of type 2 diabetes.

Pharmacological activation and genetic manipulation of cystathionine beta-synthase alter circulating levels of homocysteine and hydrogen sulfide in mice

Hydrogen sulfide (H(2)S) is a recently discovered gasotransmitter found in mammalian tissues and blood. Treatment with H(2)S donor molecules has shown promising results in preclinical models of inflammatory and cardiovascular diseases. Augmentation of H(2)S levels thus holds promise as a novel therapeutic approach for treatment of disease in man. Cystathionine β-synthase (CBS) has been shown to catalyze H(2)S production in vitro. CBS enzyme activity is allosterically regulated by the endogenous activator S-adenosyl methionine. This mode of regulation suggests the possibility for designing a small molecule activator of CBS to enhance H(2)S production. This hypothesis, however, has not been directly tested in vivo. We show here that CBS contributes significantly to endogenous H(2)S production in mice: adenovirus mediated over expression of CBS in the liver significantly increased circulating levels of H(2)S, whereas CBS deficiency resulted in reduced levels. We demonstrate that CBS enzyme from endogenous sources can be activated by S-adenosyl methionine to a greater extent compared to recombinant enzyme, suggesting greater potential for activation than previously anticipated. Importantly, we show that circulating H(2)S levels are increased by pharmacological activation of CBS in vivo; i.e. in the presence of the endogenous activator. Together, our data demonstrate that CBS activity partially regulates endogenous H(2)S in mice, and suggest that pharmacological activation of CBS is a promising approach for enhancing endogenous production of H(2)S for the treatment of cardiovascular and other diseases.

AAV8-Mediated Long-Term Expression of Human LCAT Significantly Improves Lipid Profiles in hCETP;Ldlr+/− Mice

Lecithin:cholesterol acyltransferase (LCAT) is the key circulating enzyme responsible for high-density lipoprotein (HDL) cholesterol esterification, HDL maturation, and potentially reverse cholesterol transport. To further explore LCAT’s mechanism of action on lipoprotein metabolism, we employed adeno-associated viral vector (AAV) serotype 8 to achieve long-term (32-week) high level expression of human LCAT in hCETP;Ldlr+/− mice, and characterized the lipid profiles in detail. The mice had a marked increase in HDL cholesterol, HDL particle size, and significant reduction in low-density lipoprotein (LDL) cholesterol, plasma triglycerides, and plasma apoB. Plasma LCAT activity significantly increased with humanized substrate specificity. HDL cholesteryl esters increased in a fashion that fits human LCAT specificity. HDL phosphatidylcholines trended toward decrease, with no change observed for HDL lysophosphatidylcholines. Triglycerides reduction appeared to reside in all lipoprotein particles (very low-density lipoprotein (VLDL), LDL, and HDL), with HDL triglycerides composition highly reflective of VLDL, suggesting that changes in HDL triglycerides were primarily driven by the altered triglycerides metabolism in VLDL. In summary, in this human-like model for lipoprotein metabolism, AAV8-mediated overexpression of human LCAT resulted in profound changes in plasma lipid profiles. Detailed lipid analyses in the lipoprotein particles suggest that LCATs beneficial effect on lipid metabolism includes not only enhanced HDL cholesterol esterification but also improved metabolism of apoB-containing particles and triglycerides. Our findings thus shed new light on LCAT’s mechanism of action and lend support to its therapeutic potential in treating dyslipidemia.

C-terminal Loop Mutations Determine Folding and SecretionProperties of PCSK9

Human genetics and pharmacologic clinical intervention demonstrate the key role of PCSK9 in cholesterol regulation. To understand the role of the C-terminal domain of PCSK9, two human mutations in this region (S462P and A522T PCSK9) have been profiled. Confirming and extending previous observations, S462P and WT PCSK9 bind to LDLR with equivalent affinity; however, while S462P PCSK9 cleavage is unaffected, its secretion is defective, and association with the ER protein-folding sensor calreticulin, increased. In a similar manner, A522T PCSK9 also exhibits defective secretion and an enhanced association with calreticulin. To assess the in vivo lipid phenotype of the S462P and A522T PCSK9 mutations, Pcsk9-/- mice were infected with AAV8’s encoding the different variants. Although liver transcript levels for all were equivalent, circulating levels of S462P PCSK9, and to a lesser degree A522T PCSK9, were reduced relative to WT PCSK9 correlating with the in vitro phenotype. Further, the extent of reduced circulating S462P or A522T PCSK9 correlated well with increases in mouse liver LDLR and reductions of LDL/ total cholesterol. When interpreted within the context of molecular modeling, it appears that the human non-synonymous polymorphisms S462P and A522T destabilize the C-terminal domain of PCSK9 impacting folding and secretion.

POTENTIATION OF TAU PATHOLOGY BY THE APOEε4 DEMENTIA RISK ALLELE

Background:The cognitive reserve (CR) induced by factors such as education have been associated to a higher capability of individuals affected by Alzheimer’s disease (AD) to face the effects of neurodegeneration, thus resulting in a longer-lasting normal clinical appearance. We investigated the association between CR and tau deposition in healthy controls and mild-cognitively impaired (MCI) patients. Methods:Data were obtained from the ADNI database (www.loni.ucla.edu/ADNI). 113 patients received tau-PET imaging using 18F-AV-1451 and 3D T1 MP-RAGE MRI imaging. This preliminary analysis was performed on a subset of 22 subjects (age: 78.765.9, male/female: 13/9): 12 healthy controls (HC) and 10 late MCI (LMCI) patients. Each MRI image was registered to the Montreal Neurological Institute (MNI) T1 MRI brain template. For voxelwise statistical analyses in SPM 12 (Function Image Laboratory, Wellcome Department of Cognitive Neurology, London, UK), PET images were co-registered and spatially normalized to the subject’s MP-RAGE image, followed by smoothing using an 8 mm isotropic Gaussian kernel. SUVR images were calculated using the cerebellar crus as reference region. We used two multiple linear regression analyses in the entire sample to predict the interaction between cognitive status (expressed by the Mental State Examination (MMSE) score and tau deposition, and CR (expressed as the number of years of education) and tau deposition. MMSE score and years of education were considered as independent variables and tau deposition as dependent variable. The effect of age and MMSE score, or the number of years of study, were covaried out for all voxels. Findings meeting a height threshold of p< 0.001, uncorrected, were considered significant. Results:Asexpected, a significant negative correlation between tau binding and MMSE score was retrieved in the lateral temporal cortex, bilaterally, and in left frontal cortex. For education, a significant positive correlation was found in the superior temporal gyrus, bilaterally. We expect this result to be further confirmed by extending the analysis to the whole dataset. Conclusions: A positive correlation between higher tau deposition and education was shown in the superior temporal gyrus, bilaterally, suggesting that additional cognitive resources associated with years of education can compensate for a higher degree of pathology in this region.

676. DNA Sequences Encoding shRNAs Can Replace Mutant ITR in scAAV Genome for Efficient Replication and Packaging and Transcribe shRNAs by pol III Promoter Activity of wt ITR for Efficient Gene Silencing

The two inverted terminal repeats (ITRs) at each end of recombinant adeno-associated virus (rAAV) genome are the only sequences inherited from wild type (Wt) viral genome. They contribute to rAAV genome replication, packaging, intracellular processing and stabilization, and integration into host genome. Previously we showed DNA sequences encoding shRNAs (shDNA) are the barriers in self-complementary AAV (scAAV) genome replication, resulting in vector genome truncations and heterogeneity. We now observe the same phenomena in the genome replication and packaging of single-stranded AAV vectors harboring shDNA. When detected on a denaturing gel, these truncated genomes were doubled in size as seen for scAAV, indicating they are intra-molecular double-stranded DNA molecules. We hypothesized DNA with hairpin structures could serve as the mutant ITR during rAAV genome replication and packaging. To test this, we replaced the mutant ITR in scAAV genome with the DNA encoding shRNA against Apob or Firefly luciferase gene. Southern blot of Hirts DNAs revealed that vector genomes flanked with shDNA and wtITR, but not with shDNA and mutant ITR, were efficiently rescued and replicated when co-transfected with adeno-helper plasmid and Rep/Cap trans-plasmid into HEK293 cells. Large scale AAV production and analyses of CsCl gradient purified rAAV genomes in both native and denaturing gels further demonstrated that hybrid shDNA-wtITR vector genomes were efficiently packaged into AAV capsid as double-stranded genomes. To test their functionalities in vivo, we intravenously injected 2×1011 GCs of scAAV9 containing shApoB-wtITR genome with an EGFP gene into adult C57 mice and harvested liver tissues 3 weeks later. Compared to regular scAAVEGFP vector, the shApoB-wtITR AAV vectors achieved comparable EGFP transduction efficacy in the liver. Interestingly, without a H1 promoter the shApob produced abundant small antisense RNA and silenced mouse ApoB gene as efficienct as conventional scAAVshRNA vectors. Southern blots of mouse liver detected linear and circularized forms of AAV genomes, thus implying that wtITR in the circularized genome may serve as a strong pol III promoter to drive shRNA expression. Currently, we are in the process of replacing Wt ITR with an artificial chimeric ITR with only rep binding element, terminal resolution sites and D sequence of authentic ITR retained. In summary, hairpin structure sequences can function as an alternative mutant ITR for production of functional scAAV. Our findings may have significant impact on AAV biology and vectorology, potentially enabling us to create artificially intelligent and programmable vector genomes with improved packaging efficacy, expanded genome sizes, modulated tissue tropism and propensity of integration, regulatable in vivo persistence and potency, etc.